CN103168676A - New Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method - Google Patents
New Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method Download PDFInfo
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Abstract
The invention priovidese a new Broussonetia papyrifera mulberry tree hybrid distant hybridization and polyploidization breeding method. The method is characterized in that an inductive hybrid fruit callus doubling distant hybridization polyploid hybrid line is established by an embryo engineering and tissue culture technique of distant hybrids through a reproduction engineering kind improvement technology. The method comprises following steps: 1, obtaining distant hybrid fruits; 2, carrying out callus induction of the hybrid fruits; 3, carrying out colchicine treatment of the callus of the hybrid fruits; 4, carrying out recovery culture of treated calluses; 5, differentiating the calluses into bud seedlings; 6, differentiating the bud seedlings into roots; 7, transplanting tissue culture plants; 8, carrying out comparative observation and selection of survival plants; and 9, carrying out asexual propagation of the selected plants to form a stable kind (line). The method solves the problems comprising slow growth,weak adaptability, unclear trunk and bad material quality of present Broussonetia papyrifera, and allows the new polyploid Broussonetia papyrifera mulberry tree hybrid having the advantages of obvious trunk, strong adaptability and good material quality to be cultivated.
Description
Technical field
The present invention relates to a kind of method of distant hybridization and the new hybrid of polyploidization seed selection paper mulberry mulberry tree, belong to the tree breeding technical field of modern agriculture.
Background technology
Paper mulberry is that Moraceae (Moraceae) paper mulberry belongs to the fallen leaves shrub, well developed root system, phloem fiber are extremely flourishing, and its adaptability is extremely strong, extremely drought-resistant, barren and damp and hot, rarely damage by disease and insect, and leaf contains abundant protein, is described as " ecological recovery pioneer ", and be widely used in feed industry, but it is wooden relatively poor, and trunk is not obvious, has limited to its application on timber market and gardens, trade.And mulberry tree is as one of important commodity trees of China, material is good, trunk is obvious, and its main value is to breed silkworms and top-grade furniture market material, but that it shows aspect disease and insect resistance is relatively poor, easily taken food by insects such as mulberry longicorns, also there is certain limitation in it on annidation.Be necessary cultivation mulberry tree and paper mulberry are carried out the breed improvement of reproduction engineering.
This method is utilized the reproduction engineering technology, break through cultivation mulberry tree and wild paper mulberry crossing barrier, distant hybridization and polyploidization by paper mulberry and cultivation mulberry tree, advantage between fully integrating both, both excellent genes are combined in distant hybridization polyploid hybrid, cultivate that high yield fast-growing, material are good, adaptable good economical new seeds.Both started new way for mulberry tree and paper mulberry breeding, also created good conditions for new hybrid is widely used in timber processing, gardens, trade, feed industry and Ecological Restoration Works.
Summary of the invention
The objective of the invention is to propose a kind of method of distant hybridization and polyploidization seed selection paper mulberry and the new hybrid of mulberry tree, by the reproduction engineering variety improving technique, embryo engineering, tissue culture technique are carried out in distant hybrid, foundation obtains allocarpy, inducement crossbreeding fruit callus doubles to form distant hybridization polyploid hybrid and is, not clear with the trunk that be used for to solve existing paper mulberry, the relatively poor and not strong problem of mulberry tree adaptability of material.
Technical scheme of the present invention is: the acquisition of a. distant hybrid fruit; B. the callus induction of allocarpy; C. the colchicine of allocarpy callus is processed; D. the renewal cultivation of callus after processing; E. the Calli Differentiation seedling that sprouts; F. buds differentiation goes out root; G. group is cultivated the transplanting of strain; H. survive comparative observation and the selection of plant; I. the asexual propagation of roguing becomes distinct variety (being).
Detailed process of the present invention is:
A. the acquisition of distant hybrid fruit
Distant hybrid is take good mulberry tree breed and wild paper mulberry as the parent, utilize the dioecious characteristic of paper mulberry mulberry tree, selecting female paper mulberry is that female parent carries out bagging, the pollen of male parent mulberry tree is collected in initial bloom stage, taking out flower pesticide dries in the drying place, after anther dehiscence, pollen shed, the flower pesticide paper using is wrapped, be placed on bottom and have in the drier of desiccant and store.When mucus occurring on the paper mulberry kind begins the extremely whole flower of flower, column cap, divide 2 times～3 times pollinations (choosing the pollen dispersed point on the column cap with mucus with writing brush), every other day allocarpy is sprayed hormone (50mg/L-100mg/lGA3+20mg/L-30mg/L NAA) after 2 weeks after pollination, obtain allocarpy;
B. the callus induction of allocarpy
Fetch allocarpy after flowing water washes down, first soak 30 min in 0.1% bromogeramine (5%) solution, then with 0.1% mercuric chloride, the 8～10min that sterilizes, then use aseptic washing 3 times～4 times.With the allocarpy after sterilization, be placed on and remove the outer fine hair of fruit on superclean bench, be cut into the 1/2-1/4 size and keep the 0.5cm-0.8cm petiole.Again with the fruit cut surface that cuts down level put in the allocarpy callus inducing medium, be placed in 20001X illumination 10hr/d, cultivate under 25 ℃ of conditions.20 days-25 days fruits expand and in the fruit center or tangent plane and milky bulk callus closely appears;
The callus inducing medium formula of described allocarpy is: MS+1.0mg/L-2.5mg/L 6-BA+0.1mg/L-0.5mg/L NAA+4% sucrose+0.75% agar, pH5.8;
C. the colchicine of allocarpy callus is processed
When allocarpy callus growth to 40 in the times of days-45 days, callus is placed in adds 500mg/L～750mg/L colchicine, the allocarpy callus that does not add agar doubles in culture fluid, be placed on greenhouse (26 ℃) shaking table, turn 100～speed of 120 rev/mins under rotating and culturing process 48hr～60hr;
The described culture medium prescription that doubles is: MS+1.0mg/L-2.5mg/L 6-BA+0.1mg/L-0.5mg/L NAA+500mg/L～750mg/L colchicine+4% sucrose, pH5.8;
D. the renewal cultivation of callus after processing
The allocarpy callus doubles to be disposed, and takes out callus through aseptic water washing and after blotting surface moisture, is inoculated on the allocarpy callus inducing medium renewal cultivation 7 days;
E. the Calli Differentiation seedling that sprouts
Callus through renewal cultivation is inoculated into differentiation cultivation in differential medium, and after 15 days-20 days, green bud appears in callus, differentiates successively the bud seedling after 25 days-35 days, and visible Cong Miao occurs;
Described differential medium formula is: MS+2.0mg/L～3.5mg/L 6-BA+0.1mg/L-0.3mg/L NAA+3% sucrose+0.75% agar, pH5.8;
F. buds differentiation goes out root
When buds differentiation grows into 2.0cm-5.0cm when high, the bud seedling is cut off near the callus place transplanted in root media, after 25 days-30 days, bud seedling base section dissolves root and forms complete tissue and cultivates plant;
Described prescription of rooting medium is: 1/2 MS+0.1mg/L-0.3mg/L NAA+1.0mg/L-2.0mg/L IBA+2% sucrose+0.75% agar, pH5.8
G. group is cultivated the transplanting of strain
When group cultivate strain grow into 8.0cm-12.0cm high, when having 1～3 roots, to organize the transplanting of cultivating strain, at first will transplant the cultivation bottle cap of plant opens, every bottle is added 10ml～15ml sterile water, be put in the laboratory of normal temperature slow seedling 2 days, and then fully washed the medium of plant root, with plantlet of transplant in the matrix of 70% vermiculite of sterilization+15% fine sand+15% garden mould, water 1/10MS macroelement solution, cover film and black net (50% shading rate); Then sprayed 1/10MS macroelement solution in every 2 days, until plant survives, more progressively opens black net and film, make the healthy and strong growth of group training seedling;
H. survive comparative observation and the selection of plant
Difference according to hybrid plant and parental plant external form, choose Glabrous above blade, the below is the strain of primary election heterozygote along the plant of vein thinly covered hair likeness in form mulberry tree, DNA with heterozygote blade and parent's blade extracts again, adopt the SSR molecular labeling to carry out sequence alignment to it, namely use same SSR primer pair hybrid and parental gene group amplification in vitro, amplified production is separated through polyacrylamide gel electrophoresis, compare hybrid and parental gene group echo sequence similarity degree, tested plant shows the mark of two mother plants and is defined as structure mulberry hybrid result; Distinguish diploid hybrid and polyploid hybrid through the root tip chromosomes inspection;
I. roguing becomes distinct variety (being) through asexual propagation
To be defined as paper mulberry mulberry tree hybrid (2X, intercepting branch when individual plant 4X) grows into 80cm-100cm, contain 3 buds as breeding stem section by every stem section, in 3 days-15 days after the beginning of spring, asexual propagation is carried out in transplanting on the seedbed, and becomes stable heterozygote paper mulberry mulberry tree breed (being).
Polyploid paper mulberry mulberry tree hybrid variety (being) through the above-mentioned steps seed selection has several excellent results:
1. utilize the allocarpy evoked callus, induction frequency is high, and can grow in medium, absorbs nutrition fast, fast growth.
2. carry out chromosome doubling with the allocarpy callus, induce effect better than whole seedling treatment effect, and it is low to produce chimeric frequency after differentiation and seedling emergence again.
3. the distant hybrid of cultivating by the reproduction engineering technology has that trunk is obvious, and material is good, and adaptability is extremely strong, extremely drought-resistant, barren and damp and hot, therefore the significant advantage of rare damage by disease and insect has potentiality, the wide market prospects that have in timber processing and top-grade furniture market;
4. after the new hybrid polyploid of edge far away, phloem fiber is flourishing, and limb is sturdy, with luxuriant foliage and spreading branches in leafy profusion both had anti-ly fracture, cover the large advantage of area, be again very good avenue tree planting, very large promotional value is arranged on the gardens, trade;
5. distant hybridization polyploid variety protein content is high, is very good feed industry raw material, and can not taken food by birds sterile phenomenon may appear and have no result in hybrid, has good economic worth and environmental protection effect.
Description of drawings
Fig. 1 is allocarpy picture of the present invention;
Fig. 2 is that allocarpy of the present invention is cultivated the milky bulk callus picture that produces;
Fig. 3 is the allocarpy Calli Differentiation of the present invention picture that sprouts;
Fig. 4 is that allocarpy Calli Differentiation of the present invention goes out clump bud picture;
Fig. 5 is that bud of the present invention differentiates root and becomes complete paper mulberry mulberry tree cross hybrid seedling picture.
Embodiment
The present invention is further described with embodiment for the below:
A. the acquisition of distant hybrid fruit
Select good mulberry tree breed E-sang2 in Mulberry Germplasm Resources storehouse, academy of agricultural sciences, Hubei Province, "Hur" mulberry No. 32 and the Chinese Academy of Agricultural Sciences's oil plant in wild paper mulberry GSf-01 be the parent, utilize the dioecious characteristic of paper mulberry mulberry tree, carry out the bagging processing take paper mulberry as female parent; Male parent mulberry tree pollen is collected in initial bloom stage, takes out flower pesticide and dries in the drying place, after anther dehiscence, pollen shed, the flower pesticide paper using is wrapped, and is placed in the drier of quicklime to store.When mucus occurring on paper mulberry GSf-01 begins the extremely whole flower of flower, column cap, minute 3 pollinations (choosing the pollen dispersed point on the column cap with mucus with writing brush) every other day spray 2 weeks of hormone liquid to allocarpy after pollination, acquisition allocarpy (attaching pictures 1);
B. the callus induction of allocarpy
Fetch allocarpy after flowing water washes down, first soak 30 min in 0.1% bromogeramine (5%) solution, then with 0.1% mercuric chloride sterilization 10min, then use aseptic washing 4 times.With the allocarpy after sterilization, be placed on and remove the outer fine hair of fruit on superclean bench, be cut into the 1/2-1/4 size and keep the 0.5cm-0.8cm petiole.Again with the fruit cut surface that cuts down level put in the allocarpy callus inducing medium, be placed in 20001X illumination 10hr/d, cultivate under 25 ℃ of conditions.20 days-25 days fruits expand and in the fruit center or tangent plane and milky callus (attaching pictures 2) closely appears; The callus inducing medium formula of described allocarpy is: MS+2.0mg/L 6-BA+0.1mg/L NAA+4% sucrose+0.75% agar, pH5.8;
C. the colchicine of allocarpy callus is processed
When allocarpy callus growth to 40 day～45 days, callus is placed in MS+2.0mg/L6-BA+0.1mg/LNAA+750mg/L colchicine+4% sucrose, pH5.8, the allocarpy callus that does not add agar doubles in culture fluid, be placed on greenhouse (26 ℃) shaking table rotating and culturing 48hr under the speed of 100 rev/mins;
D. the renewal cultivation of callus after processing
The allocarpy callus doubles to be disposed, and takes out callus after aseptic water washing and surface moisture, is inoculated on the allocarpy callus inducing medium renewal cultivation 7 days;
E. the Calli Differentiation seedling that sprouts
Callus through renewal cultivation is inoculated into MS+3.0mg/L6-BA+0.1mg/LNAA+3% sucrose+0.75% agar, and in the differential medium of pH5.8, differentiation is cultivated.After 15 days-20 days, callus occurs differentiating successively the bud seedling after green bud was attached pictures 3,25 days-35 days, and (attaching pictures 4) appears in visible Cong Miao;
F. buds differentiation goes out root
When buds differentiation grows into 2.0cm-5.0cm, the bud seedling is cut off near the callus place transplanted in root media (1/2MS+0.3mg/LNAA+1mg/L IBA+2% sucrose+0.75% agar, pH5.8) in, after 25 days-30 days, bud seedling base section dissolves root and forms complete tissue cultivation plant (attaching pictures 5);
G. group is cultivated the transplanting of strain
To grow into 8.0cm1～2.0cm high when group is cultivated strain, when having 1～3, to organize the transplanting of cultivating strain, at first will transplant the cultivation bottle cap of plant and open, every bottle is added the 15ml sterile water, is put in the laboratory of normal temperature to delay seedling 2 days, then fully wash the medium of plant root, plantlet of transplant in the matrix of 70% vermiculite of sterilizing+15% fine sand+15% garden mould, is watered 1/10MS macroelement solution, cover film and black net (50% shading rate).Then sprayed 1/10MS macroelement solution in every 2 days, until plant survives, more progressively opens black net and film, make the healthy and strong growth of group training seedling;
H. survive comparative observation and the selection of plant
Difference according to hybrid plant and parental plant external form, choose Glabrous above blade, the below is the strain of primary election heterozygote along the plant of vein thinly covered hair likeness in form mulberry tree, the DNA of heterozygote blade and parent's blade being extracted adopts the SSR molecular labeling to carry out sequence alignment to it again, amplified production is separated through 6% polyacrylamide gel electrophoresis, and what acquired results showed that tested plant shows the peculiar bands of a spectrum of Parent is defined as structure mulberry hybrid; Distinguish diploid hybrid and polyploid hybrid through the root tip chromosomes inspection;
I. roguing becomes distinct variety (being) through asexual propagation
To be defined as paper mulberry mulberry tree hybrid (2X, intercepting branch when individual plant 4X) grows into 80cm-100cm, contain 3 buds as breeding stem section by every stem section, in 3 days-15 days after the beginning of spring, asexual propagation is carried out in transplanting on the seedbed, and becomes stable paper mulberry mulberry tree hybrid variety (being).
Claims (1)
1. the method for a distant hybridization and the new hybrid of polyploidization seed selection paper mulberry mulberry tree is characterized in that Breeding Process is:
A. the acquisition of distant hybrid fruit
Distant hybrid be grow vigorous, good mulberry tree breed and wild paper mulberry without damage by disease and insect are the parent, utilize the dioecious characteristic of paper mulberry mulberry tree, selecting female paper mulberry is that female parent carries out bagging, the pollen of male parent mulberry tree is collected in initial bloom stage, take out flower pesticide and dry in the drying place, after anther dehiscence, pollen shed, the flower pesticide paper using is wrapped, be placed on bottom and have in the drier of desiccant and store; When mucus occurring on the extremely whole flower of paper mulberry kind beginning flower, column cap, divide 2 times～3 times pollinations, every other day allocarpy is sprayed the hormone of 50mg/L-100mg/LGA3+20mg/L-30mg/L NAA after pollination, after 2 weeks, the acquisition allocarpy;
B. the callus induction of allocarpy
fetch allocarpy after flowing water washes down, first soak 30 min in 0.1% bromogeramine (5%) solution, then with 0.1% mercuric chloride sterilization 8～10min, use again aseptic washing 3 times～4 times, with the allocarpy after sterilization, be placed on and remove the outer fine hair of fruit on superclean bench, be cut into the 1/2-1/4 size and keep the 0.5cm-0.8cm petiole, again with the fruit cut surface that cuts down level put in the allocarpy callus inducing medium, be placed in 20001X illumination 10hr/d, cultivate under 25 ℃ of conditions, 20 days-25 days fruits expand and in the fruit center or tangent plane and milky bulk callus closely appears,
The callus inducing medium formula of described allocarpy is: MS+1.0mg/L-2.5mg/L 6-BA+0.1mg/L-0.5mg/L NAA+4% sucrose+0.75% agar, pH5.8;
C. the colchicine of allocarpy callus is processed
When allocarpy callus growth to 40 in the times of days-45 days, callus is placed in adds 500mg/L～750mg/L colchicine, the allocarpy callus that does not add agar doubles in culture fluid, be placed on greenhouse (26 ℃) shaking table, turn 100～speed of 120 rev/mins under rotating and culturing process 48hr～60hr;
The described culture medium prescription that doubles is: MS+1.0mg/L-2.5mg/L 6-BA+0.1mg/L-0.5mg/L NAA+500mg/L～750mg/L colchicine+4% sucrose, pH5.8;
D. the renewal cultivation of callus after processing
The allocarpy callus doubles to be disposed, and takes out callus through aseptic water washing and after blotting surface moisture, is inoculated on the allocarpy callus inducing medium renewal cultivation 7 days;
E. the Calli Differentiation seedling that sprouts
Callus through renewal cultivation is inoculated into differentiation cultivation in differential medium, and after 15 days-20 days, green bud appears in callus, differentiates successively the bud seedling after 25 days-35 days, and visible Cong Miao occurs;
Described differential medium formula is: MS+2.0mg/L～3.5mg/L 6-BA+0.1mg/L-0.3mg/L NAA+3% sucrose+0.75% agar, pH5.8;
F. buds differentiation goes out root
When buds differentiation grows into 2.0cm-5.0cm when high, the bud seedling is cut off near the callus place transplanted in root media, after 25 days-30 days, bud seedling base section dissolves root and forms complete tissue and cultivates plant;
Described prescription of rooting medium is: 1/2 MS+0.1mg/L-0.3mg/L NAA+1.0mg/L-2.0mg/L IBA+2% sucrose+0.75% agar, pH5.8;
G. group is cultivated the transplanting of strain
When group cultivate strain grow into 8.0cm-12.0cm high, when having 1～3 roots, to organize the transplanting of cultivating strain, at first will transplant the cultivation bottle cap of plant opens, every bottle is added 10ml～15ml sterile water, be put in the laboratory of normal temperature slow seedling 2 days, and then fully washed the medium of plant root, with plantlet of transplant in the matrix of 70% vermiculite of sterilization+15% fine sand+15% garden mould, water 1/10MS macroelement solution, cover film and black net (50% shading rate); Then sprayed 1/10MS macroelement solution in every 2 days, until plant survives, more progressively opens black net and film, make the healthy and strong growth of group training seedling;
H. survive comparative observation and the selection of plant
difference according to hybrid plant and parental plant external form, choose Glabrous above blade, the below is the strain of primary election heterozygote along the plant of vein thinly covered hair likeness in form mulberry tree, DNA with heterozygote blade and parent's blade extracts again, adopt the SSR molecular labeling to carry out sequence alignment to it, namely use same SSR primer pair hybrid and parental gene group amplification in vitro, amplified production is separated through polyacrylamide gel electrophoresis, compare hybrid and parental gene group echo sequence similarity degree, the tested plant of result shows the mark of two mother plants and is defined as heterozygote, distinguish diploid hybrid and polyploid hybrid through the root tip chromosomes inspection,
I. roguing becomes distinct variety through asexual propagation
Intercepting branch when the individual plant that is defined as paper mulberry mulberry tree heterozygote is grown into 80cm-100cm, contain 3 buds as breeding stem section by every stem section, in 3 days-15 days after the beginning of spring, asexual propagation is carried out in transplanting on the seedbed, and becomes stable heterozygote paper mulberry mulberry tree breed.
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| CN105123511A (en) * | 2015-09-08 | 2015-12-09 | 安徽省农业科学院蚕桑研究所 | Method for breeding mulberry polyploids through chemical mutagenesis |
| CN109220800A (en) * | 2018-10-18 | 2019-01-18 | 楚雄师范学院 | A method of effectively improving hybridization paper mulberry tissue-cultured seedling rooting rate |
| CN113057098A (en) * | 2021-04-06 | 2021-07-02 | 广东省农业科学院蚕业与农产品加工研究所 | Rapid induction method of polyploid mulberry |
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